Lyda Neeleman

A Novel WRKY Transcription Factor Is Required for Induction of PR-1a Gene Expression by Salicylic Acid and Bacterial Elicitors

PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions −564 (box WK1) and −859 (box WK2). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK1 box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoter∷β-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35S∷NtWRKY12 and PR-1a∷GUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.

REPLICATION OF AN INCOMPLETE ALFALFA MOSAIC VIRUS GENOME IN PLANTS TRANSFORMED WITH VIRAL REPLICASE GENES

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1, 2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the P1-plants were able to replicate RNAs 2 and 3, that the P2-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, P1-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2.

ROLE OF ALFALFA MOSAIC VIRUS COAT PROTEIN GENE IN SYMPTOM FORMATION

On Samsun NN tobacco plants strains 425 and YSMV of alfalfa mosaic virus (AIMV) cause mild chlorosis and local necrotic lesions, respectively. DNA copies of RNA3 of both strains were transcribed in vitro into infectious RNA molecules. When the 425 and YSMV transcripts were inoculated to tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2, they induced symptoms indistinguishable from those of the corresponding parent strains. Exchange of restriction fragments between the infectious clones showed that symptom expression was determined by the coat protein gene in RNA3. The sequence of YSMV RNA3 was determined and compared with the known sequence of 425 RNA3. When the codon for Gln-29 in the coat protein of strain 425 was mutated into the Arg codon present at this position in strain YSMV, the symptoms induced by the transcript on inoculated leaves changed from chlorosis to necrosis. Genetic determinants for the systemic response were more complex.

Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5′-cap structure and the poly(A) binding protein bound to the 3′-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3′ end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3′ end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis

Replication of wild type RNA 3 of alfalfa mosaic virus (AIMV) and mutants with frameshifts in the P3 or coat protein (CP) genes was studied in protoplasts from tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2. Accumulation of viral plus and minus strand RNAs was monitored with strand-specific probes. A frameshift in the P3 gene did not change the asymmetry in plus/minus strand accumulation observed for the wild type. A frameshift early in the CP gene resulted in a 100-fold reduction in plus strand accumulation and a 3- to 10-fold increase in minus strand accumulation. A frameshift late in the CP gene caused a similar reduction in plus strand accumulation but had no effect on minus strand accumulation. This latter mutant accumulated nearly wild type levels of a truncated CP molecule. Apparently, wild type AIMV CP up-regulates plus strand accumulation and down-regulates minus strand accumulation and these two functions can be mutated separately.

Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions

ABSTRACT Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration ofNicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3′-untranslated regions (UTRs) of both RNAs deleted (R1Δ/2Δ construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Δ/2Δ construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Δ/2 (3′-UTR of RNA 1 deleted), R1/2Δ (3′-UTR of RNA 2 deleted), or R1Δ/2Δ plus R3. This demonstrates that the 3′-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3′-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3′-termini of AMV RNAs is not required for assembly of virus particles.

Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in Vivo

DNA copies of alfalfa mosaic virus (AIMV) RNA 3 were transcribed in vitro into RNA molecules with deletions in coding and noncoding sequences. The replication of these transcripts was studied in protoplasts from transgenic tobacco plants expressing DNA copies of AIMV RNAs 1 and 2. Deletions in the 5-proximal P3 gene, encoding the putative viral transport function, did not affect replication whereas deletions in the 3-proximal coat protein gene reduced replication of RNA 3 by about 100-fold. Sequences required for the synthesis in protoplasts of RNA 4, the coat protein messenger, were more extensive than the subgenomic promoter characterized previously in an in vitro replicase assay. At the 5-end of RNA 3 a sequence of 169 nucleotides was sufficient for replication whereas a sequence of 112 nucleotides was not. 3-Terminal deletions up to 133 nucleotides reduced replication to a low but significant level. Further 3-deletions abolished replication.

Complementation and recombination between Alfalfa Mosaic Virus RNA3 mutants in tobacco plants

n Abstractn n Deletions were made in an infectious cDNA clone of alfalfa mosaic virus (AIMV) RNA3 and the replication of RNA transcripts of these cDNAs was studied in tobacco plants transformed with AIMV replicase genes (P12 plants). Previously, we found that deletions in the P3 gene did not affect accumulation of RNA3 in P12 protoplasts whereas deletions in the coat protein (CP) gene reduced accumulation 100-fold (A. C. van der Kuyl, L. Neeleman, and J. F. Bol, 1991, Virology 183, 687–694). In P12 plants deletions in the P3 gene reduced accumulation by about 200-fold and accumulation of CP deletion mutants was not detectable. When P12 plants were inoculated with a mixture of P3- and CP-deletion mutants, both mutants replicated efficiently and various amounts of full-length RNA3 molecules were formed by recombination. The observation that some P3 and CP mutants did not recombine at a detectable level after several passages in P12 plants demonstrated that mutations in the AIMV P3 and CP genes can be complemented in trans.n n n

In Vivo and in Vitro Translation of the RNAS of Alfalfa Mosaic Virus

The translation of the purified RNAs of alfalfa mosaic virus, a virus with a coat protein dependent tripartite genome, was studied in different systems. On each RNA only one initiation site was found except on RNA 3. To this RNA two ribosomes could be bound in the presence of inhibitors of the protein elongation. Both in vivo and in vitro coat protein was formed under the direction of RNA 3. The discrepancy between the results of the infectivity and the translation studies, can either be due to an influence of RNA 1 and/or RNA 2 on the translation of RNA 3, or to a difference in regulation between wheat germ extracts and oocytes on one hand and tobacco and bean cells on the other hand. In the E.coli system the translation of RNA 4 can start either with acetylphenylalanine or with formylmethionine. From a comparison of the translation products we calculated that the initiation codon is located at or beyond position 40 from the 5′ terminus.

Biologically active transcripts of cloned DNA of the coat protein messenger of two plant viruses.

SummaryTo initiate infection, a mixture of the three genomic RNAs of alfalfa mosaic virus (AIMV) has to be supplemented with a small amount of coat protein or RNA 4, the subgenomic messenger for coat protein. The possibility to replace RNA 4 in the inoculum by in vitro synthesized transcripts of a cloned DNA copy of the coat protein cistron was investigated using the SP6 transcription system. Transcripts with or without the cap structure m7G(5′)ppp(5′)G were both translated in vitro in viral coat protein, but only capped transcripts yielded an infectious mixture when added to the AIMV genomic RNAs. This indicates that the cap structure is essential to the in vivo translatin of RNA 4. Similar results were obtained with RNAs transcribed in vitro from a DNA copy of the putative coat protein cistron of tobacco streak virus (TSV). re]19850822 rv]19851203 ac]19860114